Ubiquitin-mediated protein degradation generally occurs via the ubiquitin proteosome pathway. In this pathway, ubiquitin-protein ligases (e.g., E3 ligases) target selected proteins for degradation by ligating ubiquitin chains to the proteins to be degraded. Ubiquitination enables the proteins to then be recognized by a proteosome complex, which then degrades the proteins. Speckle type POZ protein (SPOP) has been implicated in the ubiquitin-mediated degradation of target proteins by serving the function of substrate recognition.
SPOP generally comprises two domains: a MATH domain and a poxvirus and zinc finger (POZ) domain, alternatively referred to as a BTB domain. The general structure of SPOP is illustrated in FIG. 1. SPOP proteins typically recruit protein substrates (e.g., SPOP substrates) to E3 ubiquitin ligases via its MATH and POZ domains. SPOP substrates typically complex with the MATH domain of SPOP proteins, while Cullin-3 E3 ubiquitin ligase interacts with the POZ domain. SPOP-mediated recruitment of SPOP substrates to Cullin-3 type E3 ubiquitin ligase facilitates E3-mediated ubiquitination of SPOP substrates, thus targeting the substrates for proteosomal degradation.
Exome sequencing of human prostate tumors revealed that a subset of prostate tumors comprise SPOP mutations (Nature Genetics 2012; 44: 685-689, hereby incorporated by reference). Altogether, SPOP mutations were found to be present in 6-15% of prostate tumors in the study (Nature Genetics 2012; 44: 685-689). Interestingly, all of the identified SPOP mutations associated with prostate tumors were within the substrate-binding pocket of SPOP (Nature Genetics 2012; 44: 685-689), indicating that such mutations may impair SPOP substrate binding and subsequent ubiquitination (Proc Natl Acad Sci USA. 2013 110: 6997-7002, hereby incorporated by reference). It was reported that SRC-3 protein was overexpressed in 38% of tumor samples of prostate cancer (Br J Cancer. 2001; 85: 1928-36, hereby incorporated by reference). Another study showed that SRC-3 expression is required for prostate cancer cell proliferation and survival, and its levels correlated with Prostate Specific Antigen (PSA). In a batch of prostate cancer samples from patients with prostectomy, tumor with high expression of SRC3 was shown to be correlated with lower recurrence free survival (Cancer Res. 2005 Sep. 1; 65(17):7976-83, hereby incorporated by reference). These studies suggest an important role of SRC-3 in prostate cancer formation and as a poor prognostic factor. Furthermore, SPOP mutations associated with prostate cancer impede the ability of SPOP to induce ubiquitin-dependent degradation of SRC-3 (Proc Natl Acad Sci USA. 2013 110: 6997-7002). SRC3 has also been shown to be amplified in breast cancer, both at the gene level and at the transcript level (Nature Reviews Clinical Oncology 2010; 7:83-89, hereby incorporated by reference). For example, in one study, 58% of breast tumor biopsies exhibit elevated SRC3 gene expression levels. Elevated SRC3 levels are also associated with a number of other cancers. In addition to breast cancer and prostate cancer, elevated SRC3 levels were demonstrated in pancreatic cancer and gastric cancer.
Mutational and expressional analysis of SPOP was done by Kim et al. (APMIS. 2013; 121: 626-33) in 45 gastric cancer, 45 colorectal cancer and 45 prostate cancer samples by single-strand conformation polymorphism (SSCP). In addition, they also analyzed SPOP protein expression in 60 gastric cancer, 60 colorectal cancer and 60 prostate cancer tumor specimens by immunohistochemistry. Three somatic missense mutations (2 in prostate cancer and one in colorectal cancer) of SPOP gene in the coding sequences (Ser14Leu, Tyr87Cys and Phe133Leu) were all located in the N-terminal substrate-binding domain. In the immunohistochemistry, SPOP protein was expressed in normal gastric, colonic and prostate epithelial cells, whereas loss of SPOP was found in 30% of gastric cancer, 20% of colorectal cancer and 37% of prostate cancer.
SRC3 also plays a critical role in breast cancer and other malignancies (Science 1997; 277: 965-8, Ann Oncol. 2013; 24: 1414). In a study reported by Burandt (Breast Cancer Res Treat. 2013 137:745-53), 2,197 breast carcinomas samples were analyzed and SRC3 overexpression by gene profiling and immunohistochemistry (IHC) was associated with tumor size, high histological grade, poor disease-specific, and overall survival. AIB1 amplification by fluorescent-in-situ hybridization (FISH) was found in 11% of the carcinomas. It was associated with high histological grade, lymph node involvement, and poor disease-specific survival (Breast Cancer Res Treat. 2013 137:745-53).
SRC3 amplification was observed in 7%, and over-expression in 40% of gastric cancer specimens (Int J Cancer. 2000; 89:217-23). SRC3 amplification usually coincided with its over-expression, and associated with poor prognosis. Interestingly, 15/86 (17.4%) cases of gastric adenocarcinomas were positive for Androgen Receptor (J. Can. Res. Clin. Onc. 2004; 130: 253-258). Patients with AR-positive tumors (AR+) had significantly worse prognosis than (AR−) patients (median survival 9 months vs 24 months, P=0.03).
One new estrogen receptor (ER) target was identified as DEK, whose expression also promotes estrogen-induced proliferation in breast cancer cells. DEK depletion enhances tamoxifen-induced cell death in ER+ breast cancer cell lines (PLoS ONE 2012; 7: e46985). DEK was previously identified as a DNA remodeling protein. DEK regulates DNA damage response and signaling repair. DEK functions as a transcriptional factor and is also reported to be an oncogene and ubiquitously expressed in nearly all organ and tumors (Nucleic Acids Res. 2011; 39: 7465-76). DEK protects tumor cells from DNA damaging agents and cell death via p53-dependent and -independent mechanisms by facilitating DNA double-strand break repair. High expression of DEK was associated with poor prognosis in gastric cancer (Diagn. Pathol. 2014; 9: 67).
Lestaurtinib is an indolocarbazole derivative that was originally found as an inhibitor of various receptor kinases, including JAK2 (IC50=0.9 nM), PDGFβ (IC50=216 nM), STAT3 (IC50=10-30 nM), TRKB (IC50=<25 nM), PRK1 (IC50=8.6 nM), and PKC (IC50=226 nM) and FLT3 (Cancer Res. 1999; 59: 2395-401, Blood. 2002; 99: 3885-91, which are hereby incorporated by reference). Due to its receptor kinase inhibitory activity, lestaurtinib was proposed as a potential therapeutic agent for prostate cancer. However, a Phase 2 human clinical trial demonstrated that treatment of prostate cancer patients with lestaurtinib failed to achieve the primary clinical endpoint of reduced PSA levels (Cancer Biol Ther. 2007; 6: 1360-7, hereby incorporated by reference). Thereafter, very limited clinical development has been ongoing for lestaurtinib.